Antisense polynucleotides contain synthetic sequences of nucleotide bases complementary to messenger RNA (mRNA or message) or the sense strand of double stranded DNA. Admixture of sense and antisense polynucleotides under appropriate conditions leads to the binding of the two molecules, or hybridization.
When these polynucleotides bind to (hybridize with) mRNA, inhibition of protein synthesis (translation) occurs. When these polynucleotides bind to double stranded DNA, inhibition of RNA synthesis (transcription) occurs. The resulting inhibition of translation and/or transcription leads to an inhibition of the synthesis of the protein encoded by the sense strand such as protein of the tissues, and more importantly here, a cellular growth factor, growth factor receptor, oncogene or protooncogene (many of which act as growth factors, receptors or mediators of signal transduction).
Previous reports have described antisense RNA inhibition of growth hormone production in rat pituitary tumor cells Paulssen et al., Biochem. Biophys. Res. Commun., 171:293-300 (1990)!, cytoplasmic Raf-1 protein kinase in NIH/3T3 cells Kolch et al., Nature, 349:426-428 (1991), and E-cadherin in highly invasive epithelial tumors of dog or mouse mammary gland origin Vleminckx et al., Cell, 66:107-119 (1991)!.
In one study, an antisense RNA complementary to the protooncogene c-myc sequence of the HL-60 promyelocytic leukemia cell line reduced c-myc expression, leading to cellular differentiation. The expression of c-myc was inhibited at both translational and transcriptional levels. The RNA--RNA duplex formed increased the levels of two proteins (74 and 110 Kd). The 74 Kd protein, acting as a negative repressor, binds to the CACCTCC repeat found in the c-myc leader sequence and inhibits c-myc mRNA transcription resulting in monocytic differentiation. Yokoyama, In: Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS, E. Wickstrom (ed), Wiley-Liss, New York, 1991, pp. 35-51.
Reports on antisense DNA have included inhibition of growth hormone production in lymphocytes Weigent et al., Endocrinology, 128:2053-2057 (1991)!, reduction of EGF receptors in human cells Yamada et al., Exp. Cell Res., 184:90-98 (1989)!, reduction of N-myc expression Schilbach et al., Biochem. Biophys. Res. Commun., 170:1242-1248 (1990); Whitesell et al., Mol. Cell. Biol., 11:1360-1371 (1991); Rosolen et al., Cancer Res., 50:6316-6322 (1990)!, inhibition of c-myc expression in human breast cancer Watson et al., Cancer Res., 51:3996-4000 (1991)! and lymphoma McManaway et al., Lancet, 335:806-811 (1990)!, inhibition of K-ras in lung cancer Mukhopadhyay et al., Cancer Res., 51:1744-1748 (1991)!, c-myb in colon cancer Melani et al., Cancer Res., 51:2897-2901 (1991)!, and ras p21 expression Chang et al., Biochemistry, 30:8283-8286 (1991)!. In the most successful instances, antisense sequences, utilizing either RNA or DNA polynucleotides, can direct partial or complete reversion of malignant phenotypes. Ledwith et al., Mol. Cell Biol., 10:1545-1555 (1990); Sklar et al., Mol. Cell. Biol., 11:3699-3710 (1991).
Of particular interest in treating human malignancies is the inhibition by antisense polynucleotides of expression of the BCR-ABL gene in chronic myelogenous leukemia. Szczylik et al., Science, 253:562-565 (1991). In this case a functional BCR-ABL gene maintained the leukemic phenotype and treatment with antisense DNA, directed towards the BCR-ABL junction, suppressed leukemic colony formation.
Whereas many antisense probes are directed to the translation initiation (AUG) site, other antisense probes have been directed towards intron excision sites of pre-mRNA. Volloch et al., Biochem. Biophys. Res. Commun., 179:1593-1599 (1991); Munroe, EMBO J., 7:2523-2532 (1988); Munroe et al., J. Biol. Chem., 266:22063-22068 (1991). In some cases, where the excision is incorrect, such an approach permits the selective targeting of malignant cells that express unique DNA sequences, while sparing their normal counterparts. Such tumor cell selectivity is not possible with conventional chemo- or radiation therapy.
Burkitt lymphoma is associated with abnormal expression of the c-myc oncogene due to a DNA translocation which joins the c-myc oncogene on chromosome 8 to either of the immunoglobulin genes encoded on chromosomes 2, 14 or 22. In some cases, as a result of an abnormal excision of intron mRNA, nascent transcripts may persist in a transformed clone. Antisense DNA synthesized complementary to these abnormal intron sequences greatly inhibited the ST486 and JD38 cell lines Rosolen et al., Cancer Res., 50:6316-6322 (1990)! and therefore selectively targeted only the cells with abnormal intron excision.
Antisense polynucleotides can also be used to identify and quantitate the number of genes and their location in particular cells. A study in human tissue utilized in situ hybridization, in both human oral cancer and normal oral epithelium, to identify the source and targeting of transforming growth factor .alpha. (TGF-.alpha.) to proliferating cells. This study utilized .sup.35 S-labeled sense and antisense riboprobes to human TGF-.alpha. and the receptor for epidermal growth facto (rEGF) mRNA. The results suggested that the eosinophils (containing TGF-.alpha. mRNA) produce and apparently deliver TGF-.alpha. to tumor sites, where mRNA for rEGF is expressed to a greater degree by dysplastic and carcinomatous epithelium (particularly in moderately to poor differentiated types). Todd et al., J. Dent. Res., 70:917-923 (1991).
Human prostate cancer tumors are often found to contain two types of cancerous cells. A first type is sensitive to (proliferates in the presence of) androgens such as the hormone testosterone. An exemplary cell line representing this human hormone-sensitive type of prostate cancer cell is the LNCap metastatic prostate adenocarcinoma cell line (ATCC CRL 1740). The second type of cancerous cells are insensitive to testosterone and are sensitive to (proliferate in the presence of) factors that these cells themselves secrete; i.e., autocrine growth factors. An exemplary cell line representing the autocrine type of human prostate cancers is the PC-3 prostate adenocarcinoma cell line (ATCC CRL 1435), or the DU 145 prostate carcinoma cell line (ATCC HTB 81).
The hormone-sensitive prostate cancer cells cause tumors that are typically slow-growing, better differentiated and are not particularly metastatic. That type of cancerous cell growth can often be arrested by androgen deprivation, such as by castration. Chemotherapeutic treatment of patients with drugs such as Lupron (leuprolide acetate) also arrests hormone-sensitive tumor growth after an initial flare (increased symptomology) in tumor growth by a mechanism that amounts to a chemically-induced castration.
Hormone-insensitive and presumably autocrine-dependent human prostate cancer cells are more aggressive and metastatic, and their metastases are usually responsible for deaths due to prostate cancers. Drugs such as Lupron are not effective in arresting growth of hormone-insensitive prostate cancer cells.
The PC-3 cell line, an established line of human prostate cancer cells, is considered an acceptable in vitro model system for studying this disease. Kaighn et al., Invest. Urol., 17:16-23 (1979). This undifferentiated cell line is hormone-insensitive and was established in 1979 from a bone marrow metastasis. PC-3 cells also secrete and respond to autocrine growth factors, including transforming growth factor-alpha (TGF-.alpha.). Hofer et al., Cancer Res., 51:2780-2785 (1991). TGF-.alpha. appears to stimulate PC-3 tumor cell growth by a complicated and poorly understood mechanism of signal transduction, that begins with the binding of TGF-.alpha. to the epidermal growth factor receptor (rEGF).
TGF-.alpha. is structurally and functionally related to epidermal growth factor (EGF) Thompson, Cancer Cells, 2:345-354 (1990)!, both of which are bound equally to rEGF because of the 35-40 percent amino acid sequence homology between the two growth factors Marquardt et al., Proc. Natl. Sci. USA, 80:4684-4688 (1983)!. The in vivo precursor of TGF-.alpha. is a 160 amino acid residue membrane-bound protein (pro-TGF-.alpha.) that is cleaved to yield a soluble compound. Massague, J. Biol. Chem., 265:21393-21396 (1990). This cleavage removes an extracellular portion comprised of 50 amino acids with a molecular weight of 6 Kd and is considered to be an important regulatory event Pandiella et al., Proc. Natl. Acad. Sci. USA, 88:1726-1730 (1990)! that can be stimulated by phorbol esters acting via protein kinase C Pandiella et al., J. Biol. Chem., 266:5769-5773 (1991)!.
The soluble TGF-.alpha. thus formed resembles EGF structurally because it has 6 characteristic cysteine residues that form 3 disulfide bonds spaced over a region of 35-40 amino acids. Davis, New Biol., 2:410-419 (1990). Both TGF-.alpha. and EGF regulate the growth (as a mitogen) and differentiation of normal and malignant human prostatic tissues. Cultured human prostatic tumor lines contain elevated levels of TGF-.alpha. mRNA and proliferate in response to TGF-.alpha.. Wilding et al., The Prostate, 15:1-12 (1989). TGF-.alpha. appears to have both autocrine and paracrine function, stimulating physiologic activities such as cell division and angiogenesis.
When induced in transgenic mice, TGF-.alpha. has produced epithelial hyperplasia and focal dysplastic changes that resembled carcinoma in situ. Sandgren et al., Cell, 61:1121-1135 (1990). It is suggested that TGF-.alpha. and EGF interactions are partly responsible for the autonomous growth of PC-3 cells. Such interactions could also account for the escape from hormone dependence in advanced human prostatic cancers. Hofer et al., Cancer Res., 51:2780-2785 (1991).
Regulation of signal transduction, mediated through ligand binding to the rEGF, has been attempted both experimentally and clinically. The monoclonal antibody MAb 425 (available from Hybritech, Inc., San Diego, Calif.), which is directed against the rEGF, blocks EGF dependent functions such as mitogenesis and phosphorylation. MAb 425 has also been shown to block the TGF-.alpha. induced secondary messengers inositol-1,4,5-triphosphate and Ca.sup.2+. Murthy et al., Biochem. Biophys. Res. Commun., 172:471-476 (1990).
The sensitivity of PC-3 cells to TGF-.alpha. acting at the rEGF is further demonstrated by the lack of PC-3 cell growth in serum free media in the presence of monoclonal antibodies directed to these regulatory proteins. Hofer et al., Cancer Res., 51:2780-2785 (1991). Clinically, the inhibition of prostatic tumor cell growth by suramin is thought to be mediated by blockage of TGF-.alpha. binding to the rEGF. Kim et al., J. Urol., 146:171-176 (1991). TGF-.alpha. expression may also be stimulated by the ras-encoded p21 oncoprotein. Aaronson et al., In: Molecular Foundations of Oncology, S. Broder (ed.), Williams and Wilkins, Baltimore, Md., pp. 27-39, 1991.
Epidermal growth factor (EGF) is a 53-residue polypeptide, and is a potent in vitro mitogen for a variety of cell types. Harris, Am. J. Kidney Dis., 17:627-630 (1991). Although the relationship of EGF to hormone (testosterone) and cellular organization (cytokeratins) is complex, EGF is implicated in the progression of prostate cancer. Fowler et al., J. Urol., 139:857-861 (1988). In addition, progression of prostate cancer from hormone dependence to independence following castration is associated with a coordinate loss of growth regulatory factors. Rubenstein et al., Clin. Physiol. Biochem., 9:47-50 (1992). It has been suggested that control of EGF following hormone independence might be therapeutically efficacious. Fowler et al., J. Urol., 139:857-861 (1988).
Both TGF-.alpha. and EGF are degraded by metalloproteases, but involve separate degradative pathways. This level of regulation could account for some differential effects encountered in various systems. Gehm et al., Endocrinology, 128:1603-1610 (1991).
The human rEGF is a membrane-bound glycoprotein of 170 kd that has been identified in human prostatic tissue and the PC-3 cell line. Hofer et al., Cancer Res., 51:2780-2785 (1991). The regulatory importance of rEGF is evidenced by the fact that the expression of mRNA for rEGF is greater in carcinomas of the prostate than that found in samples of benign prostatic hyperplasia, the greatest levels of rEGF mRNA being found in the human prostatic cell lines PC-3 and DU145. Morris et al., J. Urol., 143:1272-1275 (1990).
Following rEGF binding of either TGF-.alpha. or EGF, tyrosine kinase activity is stimulated via signal transduction mechanisms, initiating mitogenesis. Thompson et al., Cancer Surveys, 4:767-788 (1985). The number of rEGF molecules found upon the cell surface correlates to cell growth regulation. In the malignant human glioma D-298 MG Humphrey et al., Biochem. Biophys. Res. Commun., 178:1413-1420 (1991)!, it has been suggested that overexpression of the rEGF leads to enhanced binding by EGF and TGF-.alpha., resulting in increased tyrosine kinase activity. Therefore overexpression, rather than a structural alteration, may account for most increases in biologic activity. Humphrey et al., Biochem. Biophys. Res. Commun., 178:1413-1420 (1991).
This receptor is itself very important in growth regulation not only because it binds the EGF ligand (as well as TGF-.alpha.) but also because the gene for this receptor is homologus to the erb-B2 and erb-B3 protooncogenes, which, when similarly overexpressed (in adenocarcinomas of the breast, stomach, ovary, colon and salivary gland) or structurally altered (in intestinal adenocarcinoma) from its protooncogene state, stimulates uncontrolled growth Aaronson et al., In: Molecular Foundations of Oncology, S. Broder (ed.), Williams and Wilkins, Baltimore, Md., pp. 27-39 (1991)!, probably by excessive stimulation of tyrosine kinase activity.
It would therefore be beneficial if a chemotherapeutic treatment could be devised that could arrest the growth and kill autocrine-sensitive or dependent prostate cancer cells such as those of the PC-3 cell line that are no longer susceptible to androgen deprivation therapy. The invention discussed hereinafter describes such a chemotherapeutic treatment and treating agents.